Investigations of the determinants of ATP binding in the active site of 5,10‐methenyltetrahydrofolate synthetase form Mycoplasma pneumonia

The pathways for the synthesis of tetrahydrofolates form the core of one carbon metabolism. As such, these pathways are a source of targets in the treatment of cancer. 5‐formyltetrahydrofolate (5‐formylTHF) is administered as a rescue agent or as an enhancer in these treatments. The only enzyme known to use 5‐formylTHF as a substrate is 5,10‐methenyltetrahydrofolate synthetase (MTHFS). This enzyme catalyzes the conversion of 5‐formylTHF to 5,10‐methenyltetrahydrofolate in conjunction with the hydrolysis of ATP to ADP. To better understand the function of MTHFS, the determinants of ATP binding in the active site were investigated through site‐directed mutagenesis followed by circular dichroism spectroscopy and kinetics. Replacement of aspartates at positions 124 and 154 with alanine (mutants D124A and D154A) resulted in no detectable activity while mutants K3A, R7A, R125A, and W153A demonstrated substantially decreased affinity for ATP. Mutants D81A and D151A had ATP affinity similar to wild type. Circular dichroism data confirmed that all mutants were folded. These results are consistent with the co‐crystal structure of MTHFS with 5‐formylTHF, ADP, and phosphate (Proteins. 61: 433).